Summary of work: Breast cancer incidence and mortality increase dramatically after the age of 65 and are influenced by reproductive factors, the presence of estrogen receptors (ER), and perhaps dietary factors. We have developed improved methods to grow lines of ER positive (ER+) breast tumor cells arising spontaneously in aged female rats. These cells progress to hormone-independent malignant disease in vivo and ER negative (ER-) cells can be isolated allowing the investigation of the genetic events underlying the development of these cancers. Previous studies using cell fusion of ER+ and ER- cells showed that probable loss of tumor suppressor gene (s) is important in progression. In further studies, the properties of ER+ and ER- negative cells were compared. ER+ cells were less invasive, more sensitive to programmed cell death, and expressed a different profile of proteolytic activities than ER- cells. We have found that regulation of the urokinase plasminogen activator (uPA) gene in these ER+ cells was under hormonal control, whereas ER- cells did not express uPA. Hormones such as prolactin and hCG stimulated uPA production while progesterone and dexamethasone inhibited it. Matrix molecules such as fibronectin and collagen I also inhibited uPA production while laminin and collagen IV did not, suggesting that specific integrin interactions were important in signalling. In addition, cell-cell binding, perhaps through E-cadherin sites, was also essential to stimulate uPA expression. These studies indicate that precise regulation of uPA activity may be involved in matrix remodeling in hormone-responsive cells and not in invasion as it is in hormone-independent cells. Studies of apoptosis sensitivity in this progression model showed that ER- cells became resistant to apoptosis induced by the chemotherapeutic drug doxorubicin. ER+ cells, however, expressed the putative tumor suppressor gene p73 and activated a caspase-3-like activity. In these cells apoptosis was associated with decreased p21 but elevated Bax expression. p53 expression was undetectable. Since progression to hormone independence results in loss of uPA regulation and leads to acquisition of apoptosis resistance, identification of the putative suppressor gene(s) controlling these phenotypes may lead to therapeutic strategies to prevent breast tumor progression or to treat the malignant disease.